Antonie van Leeuwenhoek (2014) 105:135–141 DOI 10.1007/s10482-013-0061-8
ORIGINAL PAPER
Salinarubrum litoreum gen. nov., sp. nov.: a new member of the family Halobacteriaceae isolated from Chinese marine solar salterns Heng-Lin Cui • Xing-Xing Qiu
Received: 26 August 2013 / Accepted: 17 October 2013 / Published online: 25 October 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Three halophilic archaeal strains, XD46T, YJ-63-S1 and ZS-1-H, were isolated from three Chinese marine solar salterns. All were observed to have pleomorphic cells that lysed in distilled water, stained Gram-negative and formed red-pigmented colonies. They were found to grow optimally at 37 °C, at pH 7.0 and in the presence of 2.6 M NaCl and 0.05 M Mg2?. The major polar lipids were identified as those typical for members of the Halobacteriaceae but also included major glycolipids chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-1), mannosyl glucosyl diether (DGD-1) and two unidentified ones. The 16S rRNA gene sequences of the three strains were 99.8–100 % identical, showing most similarity to sequences of members of the family Halobacteriaceae, and clustering together as a distinct clade in phylogenetic tree reconstructions. The rpoB0 gene similarities between the three strains were 98.7–100 % and lower to the sequences of other halobacteria. Their DNA G?C contents were determined to be 65.1–65.5 mol%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strains XD46T (=CGMCC 1.12237T =
Electronic supplementary material The online version of this article (doi:10.1007/s10482-013-0061-8) contains supplementary material, which is available to authorized users. H.-L. Cui (&) X.-X. Qiu School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Jingkou District, Zhenjiang 212013, People’s Republic of China e-mail: [email protected]
JCM 18649T), YJ-63-S1 (=CGMCC 1.12574) and ZS1-H (=CGMCC 1.12544) represent a novel species in a new genus within the family Halobacteriaceae, for which the name Salinarubrum litoreum gen. nov., sp. nov. is proposed. Keywords Salinarubrum litoreum gen. nov., sp. nov. Halophilic archaea Marine solar saltern
Introduction Marine solar salterns are common artificial habitats for diverse halophilic archaea, members of the family Halobacteriaceae within the order Halobacteriales (Oren 2006). In recent years, from this kind of hypersaline environment, more and more remarkable isolates representing novel genera have been cultivated, described and proposed, such as Halonotius pteroides (Burns et al. 2010), Halogranum rubrum, Halopelagius inordinatus, Halorussus rarus, Halolamina pelagica, Halobellus clavatus, Halorientalis regularis, Halorubellus salinus (Cui et al. 2010a, b, c, 2011a, b, 2012), Natronoarchaeum mannanilyticum, Salarchaeum japonicum (Shimane et al. 2010, 2011), Halarchaeum acidiphilum (Minegishi et al. 2010a), Halomarina oriensis (Inoue et al. 2011), Halohasta litorea (Mou et al. 2012) and Halomicroarcula pellucida (Echigo et al. 2013). This rapid expansion in new taxa indicates that the members of family
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Halobacteriaceae are more diverse than was previously recognized. During our surveys on halophilic archaeal diversity of marine solar salterns of Eastern and Southern China, three haloarchaeal strains, XD46T, YJ-63-S1 and ZS-1-H, showing high similarity of 16S rRNA gene sequence, were isolated from three different marine solar salterns. In this study, we characterize these three strains and propose they represent a novel species of a novel genus, Salinarubrum.
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to the proposed minimal standards for description of new taxa in the order Halobacteriales (Oren et al. 1997). The type strains Halorussus rarus TBN4T, Halalkalicoccus jeotgali JCM 14584T, Halorubellus salinus GX3T, Haladaptatus paucihalophilus JCM 13897T were selected as reference strains in phenotypic tests. These reference strains were obtained from China General Microbiological Culture Collection and Japan Collection of Microorganisms. Chemotaxonomic characterization
Materials and methods Isolation and cultivation of halophilic archaeal strains Strain XD46T was isolated from a surface brine sample taken from Xidi marine solar saltern, at Xiamen, Fujian Province, China (24°330 5100 N, 118°180 2100 E; elevation, sea level) in 2010. Strain YJ-63-S1 was isolated from a sediment sampling from Yangjiang marine solar saltern, at Yangjiang, Guangxi Province, China (21°310 4800 N, 111°280 500 E; elevation, sea level) in 2012. Strain ZS-1-H was isolated from the sediment of Zhoushan marine solar saltern, at Zhoushan, Zhejiang Province, China (29°560 5600 N, 122°200 2000 E; elevation, sea level) in 2012. The neutral oligotrophic haloarchaeal medium (NOM) was used for the isolation procedure, and contained the following ingredients (g/L): yeast extract (Oxoid) 0.05, fish peptone (Sinopharm Chemical Reagent Co., Ltd.) 0.25, sodium pyruvate 1.0, KCl 5.4, K2HPO4 0.3, CaCl2 0.25, NH4Cl 0.25, MgSO47H2O 26.8, MgCl26H2O 23.0, NaCl 184.0 (pH adjusted to 7.0–7.2 with 1 M NaOH solution). The medium was solidified with 2.0 % agar. The strains were routinely grown aerobically at 37 °C in NOM-3 medium (NOM series medium) with the following modifications (g/L): yeast extract 1.0, fish peptone 0.25, sodium formate 0.25, sodium acetate 0.25, sodium lactate 0.25 and sodium pyruvate 0.25. Phenotypic determination Determination of morphology and growth characteristics, nutrition, miscellaneous biochemical tests and sensitivity to antimicrobial agents were performed for all species in the same basic medium, NOM, according
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Polar lipids were extracted using a chloroform/methanol system and analysed using one-and two-dimensional TLC, as described previously (Cui et al. 2010b). Merck silica gel 60 F254 aluminium-backed thin-layer plates were used for TLC analyses. In two-dimensional TLC, the first solvent was chloroform–methanol–water (65:25:4, by vol.) and the second solvent was chloroform–methanol–acetic acid–water (80:12:15:4, by vol.). The latter solvent mixture was also used in onedimensional TLC. Two specific detection spray reagents, phosphate stain reagent for phospholipids and a-naphthol strain for glycolipids, were used. The general detection reagent, sulfuric acid–ethanol (1:2, by vol.), was also used to detect total polar lipids. The presence of phospholipids and glycolipids on the twodimensional TLC was confirmed by comparing with one-dimensional TLC on which the polar lipid profiles of reference strains were developed. Phylogenetic and genomic analysis Genomic DNA from halophilic archaeal strains was prepared as described previously (Cui et al. 2011c). The 16S rRNA genes were amplified, cloned and sequenced according to the previous protocol (Cui et al. 2009). PCR-mediated amplification and sequencing of the rpoB0 genes were performed as described previously (Minegishi et al. 2010b). Multiple sequence alignments were performed using the ClustalW program integrated in the MEGA 5 software (http://www.megasoftware. net/). Phylogenetic trees were reconstructed using neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) algorithms in the MEGA 5 software (Tamura et al. 2011). Gene sequence similarity among halophilic archaea was calculated using the Pairwise-Distance computing function of MEGA 5. The DNA G?C content was determined from
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the mid-point value (Tm) of the thermal denaturation method (Marmur and Doty 1962) at 260 nm with a Beckman-Coulter DU800TM spectrophotometer equipped with a high-performance temperature controller. Halomicrobium mukohataei JCM 9738T was selected as reference strain for these analyses, and the formula G?C mol% unknown strain = G?C mol% reference strain ? 2.08 9 (Tm unknown strain - Tm reference strain) was used to calculate the G?C content from the known Tm value (Owen and Pitcher 1985).
Results and discussion Cells of strains XD46T, YJ-63-S1 and ZS-1-H were observed to be motile and pleomorphic when grown in NOM-3 liquid medium (Supplementary Fig. S1). They were observed to stain Gram-negative and their colonies were observed to be red-pigmented. The three strains were found to be able to grow at 20–50 °C (optimum 37 °C), in the presence of 0.9–4.8 M NaCl (optimum 2.6 M NaCl), with 0.005–1.0 M MgCl2 (optimum 0.05 M MgCl2) and at pH 5.0–9.0 (optimum pH 7.0). The cells of all isolates were observed to lyse in distilled water and the minimum NaCl concentration that prevented cell lysis was 50 g/L. The three strains did not grow under anaerobic conditions using nitrate, DMSO or L-arginine, but nitrate reduction to nitrite was observed. They were found to produce H2S from sodium thiosulfate but did not produce indole from tryptophan. They hydrolyzed gelatin, Tween 80 and casein, but did not hydrolyze starch. The main phenotypic characteristics differentiating strains XD46T, YJ63-S1 and ZS-1-H from the related members of family Halobacteriaceae were: cell motility, cell lysis in distilled water, optimum NaCl for growth, the growth requirement for Mg2?, optimum pH, utilization of specific carbon sources, indole formation, starch, casein gelatin, Tween 80 and hydrolysis (Table 1). More detailed results of phenotypic tests and nutritional features of these strains are given in the species descriptions. The major polar lipids of the three strains were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, two major glycolipids chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-1) and mannosyl glucosyl diether (DGD-1), respectively. Three unidentified lipids were also detected (Supplementary Fig. S2).
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The polar lipid profile set strains XD46T, YJ-63-S1 and ZS-1-H apart from members of the genus Halorussus which contain sulfated galactosyl mannosyl glucosyl diether (S-TGD-1), S-DGD-1, galactosyl mannosyl glucosyl diether, DGD-1 and an unknown diglycosyl diether (Cui et al. 2010b), members of the genus Halorubellus which contain S-TGD-1, S-DGD1, DGD-1 and an unidentified lipid (Cui et al. 2012), and members of the Halalkalicoccus which contain trace unidentified glycolipids (Roh et al. 2007) (Supplementary Fig. S2). Eight complete 16S rRNA gene sequences of each strain were obtained (1,475 bp in length), and these indicated that each strain has a single 16S rRNA gene sequence. They shared 99.8–100 % sequence identities, and all three strains showed low levels of 16S rRNA gene sequence similarity to other members of the family Halobacteriaceae. The closest related recognized species were Halalkalicoccus jeotgali (92.3–92.4 % similarity), Halalkalicoccus tibetensis (92.2–92.3 % similarity), Halorussus rarus (91.6–91.9 % similarity) and Halorubellus salinus (90.4–91.9 % similarity). Phylogenetic tree reconstructions using the NJ algorithm revealed that strains XD46T, YJ-63-S1 and ZS-1-H tightly clustered with each other forming a distinct clade (Fig. 1a), separate from other recognized genera of the Halobacteriaceae. The closest branching species to this clade was Halorussus rarus TBN4T. Their phylogenetic position was also confirmed in other trees generated using the MP and ML algorithms (data not shown). The rpoB0 genes of all the three strains were sequenced and found to be identical in length (1,827 bp) and their sequence identities were 98.7–100 %. The most closely similar sequence was the corresponding gene of Halorussus rarus TBN4T, but the level of similarity to the three isolates was much lower (85.8–85.9 %). In phylogenetic tree reconstructions using rpoB0 gene sequences, strains XD46T, YJ-63-S1 and ZS-1-H clustered tightly with each other, forming a distinct clade, this clade branched nearby members of Halalkalicoccus and Halorussus (Fig. 1b). The phylogenetic position was also confirmed in trees generated using the MP and ML algorithms (data not shown). The DNA G?C content of strains XD46T, YJ-63S1 and ZS-1-H was determined to be 65.5, 65.1 and 65.1 mol%, respectively. These values are higher than those of members of the genera Haladaptatus (54.0–59.2 mol%) (Cui et al. 2010d) and
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Table 1 Differential characteristics between strain XD46T, strain YJ-63-S, strain ZS-1-H and closely related members of the family Halobacteriaceae Characteristic
1
2
3
4
5
6
7
Motility
?
?
?
?
±
–
?
Cells lyse in distilled water
?
?
?
?
–
–
?
Optimum NaCl (M)
2.6
2.6
2.6
2.1
2.6-3.4
2.6-3.4
3.1
Mg2? required
?
–
–
–
?
–
–
Optimum pH
7.0
7.0
7.0
7.0
7.0
7.0/9.0
7.0
D-mannose
?
?
?
?
±
–
–
D-galactose
?
?
?
?
±
–
±
D-fructose
–
–
–
–
±
?
–
Maltose
–
–
–
?
±
?
–
Lactose
–
–
–
?
±
?
– –
Utilization of:
Indole formation
–
–
–
?
?
±
Starch hydrolysis
–
–
–
?
±
–
–
Gelatin hydrolysis
?
?
?
?
?
–
?
Casein hydrolysis Tween 80 hydrolysis
? ?
? ?
? ?
? ?
± ?
– –
? –
Presence of PGS
–
–
–
?
?
–
–
G?C content (mol%)
65.5
65.1
65.1
65.4-66.1
54.0-59.2
61.5-63.2
67.2-67.3
Taxa: 1, XD46T; 2, YJ-63-S; 3, ZS-1-H; 4, Halorussus; 5, Haladaptatus; 6, Halalkalicoccus; 7, Halorubellus. Symbols: ?, positive; –, negative. All data from the present study
Halalkalicoccus (61.5–63.2 mol%) (Roh et al. 2007), lower than those of members of the genus Halorubellus (67.2–67.3 mol%) (Cui et al. 2012) and close to the values for members of the genus Halorussus (65.4–66.1 mol%) (Cui et al. 2010b). This polyphasic taxonomic study provides evidence that strains XD46T, YJ-63-S1 and ZS-1-H represent a novel species of a new genus within the family Halobacteriaceae, for which the name Salinarubrum litoreum gen. nov., sp. nov. is proposed. The type strain is XD46T (= CGMCC 1.12237T = JCM 18649T) and the reference strains are YJ-63-S1 (= CGMCC 1.12574) and ZS-1-H (= CGMCC 1.12544). Characteristics that distinguish strains XD46T, YJ-63-S1 and ZS-1-H from other genera within the family Halobacteriaceae are shown in Table 1.
Cells lyse in distilled water. Catalase test is positive and the oxidase test is negative. Extremely halophilic, with growth occurring in media containing 0.9–4.8 M NaCl; most strains grow best at 2.6 M NaCl. The optimum magnesium concentration is 0.05 M. Temperatures between 20 and 50 °C and pH between 5.0 and 9.0 may support growth. Sugars are metabolized, in some cases with the formation of acids. The polar lipids are phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, two major glycolipids chromatographically identical to S-DGD-1 (sulfated mannosyl glucosyl diether) and DGD-1 (mannosyl glucosyl diether), respectively. Three unidentified lipids are also present. The genomic DNA G?C content is between 65.1 and 65.5 mol%. Isolated from marine solar salterns. The type species is Salinarubrum litoreum. Recommended three-letter abbreviation: Srr.
Description of Salinarubrum gen. nov. Description of Salinarubrum litoreum sp. nov. Salinarubrum (Sa.li.na.ru’brum. L. fem. pl. n. salinae salterns, salt works; L. neut. adj. rubrum red; M.L. neut. n. Salinarubrum the red archaeon from salt works). Cells are pleomorphic under optimal growth conditions, stain Gram-negative and are aerobic heterotrophs.
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Salinarubrum litoreum (li.to’re.um. L. neut. adj. litoreum, of or belonging to the sea-shore). Cells are motile, pleomorphic-shaped under optimal growth conditions and stain Gram-negative. Colonies
Antonie van Leeuwenhoek (2014) 105:135–141
a
139 Haladaptatus litoreus RO1-28T (EU887285)
100
Haladaptatus paucihalophilus JCM 13897T (HM159609)
57
Halorubellus salinus GX3T rrnA (GU951429) Halorubellus salinus GX3T rrnB (HQ236376)
100
Halorussus rarus TBN4T (GQ282618)
0.02
Strain XD46T (JQ237118)
72 84
100
Strain YJ-63-S (KC918824)
65 Strain
ZS-1-H (KC918825)
Halalkalicoccus jeotgali B3T (NC_014297)
100
Halalkalicoccus tibetensis DS12T (AF435112) Halococcus morrhuae ATCC 17082T (X00662)
98
Halococcus salifodinae DSM 8989T (AB004877)
100
Natronomonas pharaonis DSM 2160T (NC_007426) Methanospirillum hungatei JF-1T (NC_007796)
b
100 100
Strain YJ-63-S (KF316330) Strain ZS-1-H (KF316331) Strain XD46T (KF316332)
63
Halalkalicoccus jeotgali JCM 14584T (AB477140) 100
0.05
Halalkalicoccus tibetensis JCM 11890T (AB477141) Halorussus rarus TBN4T (JN120805)
52
Halorubellus salinus GX3T (JN164652) Haladaptatus litoreus RO1-28T (JN315801)
90
100
Haladaptatus paucihalophilus JCM 13897T (AB477139) Halococcus morrhuae JCM 8876T (AB477157)
96
Halococcus salifodinae JCM 9578T (AB477160) Natronomonas pharaonis DSM 2160T (NC_007426) Methanospirillum hungatei JF-1T (NC_007796)
Fig. 1 Neighbour-joining phylogenetic tree reconstructions based on 16S rRNA gene (a) and rpoB0 gene (b) sequences, showing the relationships between the isolates and related members within the family Halobacteriaceae. Bootstrap values
(%) are based on 1,000 replicates and are shown for branches with more 50 % bootstrap support. Bar represents expected changes per site
on agar plates containing 2.6 M NaCl are red, elevated and round. Chemoorganotrophic and aerobic. Growth occurs at 20–50 °C (optimum 37 °C), at 0.9–4.8 M NaCl (optimum 2.6 M), at 0.005–1 M MgCl2 (optimum 0.05 M) and at pH 5.0–9.0 (optimum pH 7.0). Cells lyse in distilled water and the minimal NaCl concentration to prevent cell lysis is 50 g/L. Catalasepositive and oxidase-negative. Does not grow under anaerobic conditions with nitrate, arginine or DMSO. Nitrate reduction to nitrite is observed but gas formation from nitrate is not. H2S is produced from sodium thiosulfate. Indole formation is negative. Hydrolyzes casein, gelatin and Tween 80 but does not hydrolyze
starch. The following substrates are utilized as single carbon and energy sources for growth: citrate, fumarate, D-glucose, glycerol, DL-lactate, L-malate, pyruvate, Dsorbitol and sucrose. L-glutamate is utilized as single carbon, nitrogen or energy sources for growth. No growth occurs on acetate, L-alanine, L-arginine, Laspartate, D-fructose, D-galactose, glycine, lactose, Llysine, maltose, D-mannitol, D-mannose, L-ornithine, Dribose, L-sorbose, starch, succinate or D-xylose. Acid is produced from D-glucose and sucrose. Sensitive to the following antimicrobial compounds (lg per disc, unless otherwise indicated): bacitracin (0.04 IU per disc), ciprofloxacin (5), mycostatin (100), nitrofurantoin
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(300), norfloxacin (10) and novobiocin (30). Resistant to the following antimicrobial compounds: ampicillin (10), chloramphenicol (30), erythromycin (15), gentamicin (10), kanamycin (30), nalidixic acid (30), neomycin (30), penicillin G (10 IU per disc), rifampin (5), streptomycin (10), tetracycline (30), trimethoprim (5) and vancomycin (30). The polar lipids are phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, two major glycolipids chromatographically identical to S-DGD-1 and DGD-1, respectively. Three unidentified lipids are also detected. The DNA G?C content of strain XD46T is 65.5 mol% (Tm). The type strain is XD46T (=CGMCC 1.12237T = JCM 18649T) and was isolated from Xidi marine solar saltern at Xiamen, Fujian Province, China. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains XD46T, YJ63-S1 and ZS-1-H are JQ237118, KC918824 and KC918825 respectively. Those for the rpoB0 gene sequences of strains XD46T, YJ-63-S1 and ZS-1-H are KF316330, KF316331 and KF316332, respectively. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31370054), the grant from China Ocean Mineral Resources R&D Association (COMRA) Special Foundation (DY125-15-R-03), the Qinglan Project of Jiangsu Province and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
References Burns DG, Janssen PH, Itoh T, Kamekura M, Echigo A, DyallSmith ML (2010) Halonotius pteroides gen. nov., sp. nov., an extremely halophilic archaeon recovered from a saltern crystallizer in Southern Australia. Int J Syst Evol Microbiol 60:1196–1199 Cui H-L, Zhou P-J, Oren A, Liu S-J (2009) Intraspecific polymorphism of 16S rRNA genes in two halophilic archaeal genera, Haloarcula and Halomicrobium. Extremophiles 13:31–37 Cui H-L, Gao X, Sun F-F, Dong Y, Xu X-W, Zhou Y-G, Liu H-C, Oren A, Zhou P-J (2010a) Halogranum rubrum gen. nov., sp. nov., a halophilic archaeon isolated from a marine solar saltern. Int J Syst Evol Microbiol 60:1366–1371 Cui H-L, Gao X, Yang X, Xu X-W (2010b) Halorussus rarus gen. nov., sp. nov., a new member of the family Halobacteriaceae isolated from a marine solar saltern. Extremophiles 14:493–499 Cui H-L, Li X-Y, Gao X, Xu X-W, Zhou Y-G, Liu H-C, Oren A, Zhou P-J (2010c) Halopelagius inordinatus gen. nov., sp. nov., a new member of the family Halobacteriaceae isolated from a marine solar saltern. Int J Syst Evol Microbiol 60:2089–2093
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Antonie van Leeuwenhoek (2014) 105:135–141 Cui H-L, Sun F-F, Gao X, Dong Y, Xu X-W, Zhou Y-G, Liu H-C, Oren A, Zhou P-J (2010d) Haladaptatus litoreus sp. nov., an extremely halophilic archaeon from a marine solar saltern, and emended description of the genus Haladaptatus. Int J Syst Evol Microbiol 60:1085–1089 Cui H-L, Gao X, Yang X, Xu X-W (2011a) Halolamina pelagica gen. nov., sp. nov., a new member of the family Halobacteriaceae. Int J Syst Evol Microbiol 61:1617–1621 Cui H-L, Yang X, Gao X, Xu X-W (2011b) Halobellus clavatus gen. nov., sp. nov. and Halorientalis regularis gen. nov., sp. nov., two new members of the family Halobacteriaceae. Int J Syst Evol Microbiol 61:2682–2689 Cui H-L, Yang X, Mou Y-Z (2011c) Salinarchaeum laminariae gen. nov., sp. nov.: a new member of the family Halobacteriaceae isolated from salted brown alga Laminaria. Extremophiles 15:625–631 Cui H-L, Mou Y-Z, Yang X, Zhou Y-G, Liu H-C, Zhou P-J (2012) Halorubellus salinus gen. nov., sp. nov. and Halorubellus litoreus sp. nov., novel halophilic archaea isolated from a marine solar saltern. Syst Appl Microbiol 35:30–34 Echigo A, Minegishi H, Shimane Y, Kamekura M, Itoh T, Usami R (2013) Halomicroarcula pellucida gen. nov., sp. nov., a non-pigmented, transparent-colony-forming, halophilic archaeon isolated from solar salt. Int J Syst Evol Microbiol 63:3556–3562 Inoue K, Itoh T, Ohkuma M, Kogure K (2011) Halomarina oriensis gen. nov., sp. nov., a halophilic archaeon isolated from seawater aquarium. Int J Syst Evol Microbiol 61:942–946 Marmur J, Doty P (1962) Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5:109–118 Minegishi H, Echigo A, Nagaoka S, Kamekura M, Usami R (2010a) Halarchaeum acidiphilum gen. nov., sp. nov., a moderately acidophilic haloarchaeon isolated from commercial solar salt. Int J Syst Evol Microbiol 60:2513–2516 Minegishi H, Kamekura M, Itoh T, Echigo A, Usami R, Hashimoto T (2010b) Further refinement of Halobacteriaceae phylogeny based on the full-length RNA polymerase subunit B (rpoB0 ) gene. Int J Syst Evol Microbiol 60:2398–2408 Mou Y-Z, Qiu X-X, Zhao M-L, Cui H-L, Oh D, Dyall-Smith ML (2012) Halohasta litorea gen. nov. sp. nov., and Halohasta litchfieldiae sp. nov., isolated from the Daliang aquaculture farm, China and from Deep Lake, Antarctica, respectively. Extremophiles 16:895–901 Oren A (2006) The order Halobacteriales. In: Dworkin M, Falkow S, Rosenberg E, Schleifer KH, Stackebrandt E (eds) The prokaryotes, vol 3, 3rd. edn. Springer, New York, pp 113–164 Oren A, Ventosa A, Grant WD (1997) Proposed minimal standards for description of new taxa in the order Halobacteriales. Int J Syst Bacteriol 47:233–238 Owen RJ, Pitcher D (1985) Current methods for estimating DNA base composition and levels of DNA–DNA hybridization. In: Goodfellow M, Minnikin DE (eds) Chemical methods in bacterial systematics. Academic Press, London, pp 67–93 Roh SW, Nam YD, Chang HW, Sung Y, Kim KH, Lee HJ, Oh HM, Bae JW (2007) Halalkalicoccus jeotgali sp. nov., a halophilic archaeon from shrimp jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 57:2296–2298
Antonie van Leeuwenhoek (2014) 105:135–141 Shimane Y, Hatada Y, Minegishi H, Mizuki T, Echigo A, Miyazaki M, Ohta Y, Usami R, Grant WD, Horikoshi K (2010) Natronoarchaeum mannanilyticum gen. nov., sp. nov., an aerobic, extremely halophilic member of the Archaea isolated from commercial salt made in Niigata Japan. Int J Syst Evol Microbiol 60:2529–2534 Shimane Y, Hatada Y, Minegishi H, Echigo A, Nagaoka S, Miyazaki M, Ohta Y, Maruyama T, Usami R, Grant WD,
141 Horikoshi K (2011) Salarchaeum japonicum gen. nov., sp. nov., an aerobic, extremely halophilic member of the Archaea isolated from commercial salt made in Okinawa Japan. Int J Syst Evol Microbiol 61:2006–2010 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739
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ORIGINAL PAPER
Salinarubrum litoreum gen. nov., sp. nov.: a new member of the family Halobacteriaceae isolated from Chinese marine solar salterns Heng-Lin Cui • Xing-Xing Qiu
Received: 26 August 2013 / Accepted: 17 October 2013 / Published online: 25 October 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Three halophilic archaeal strains, XD46T, YJ-63-S1 and ZS-1-H, were isolated from three Chinese marine solar salterns. All were observed to have pleomorphic cells that lysed in distilled water, stained Gram-negative and formed red-pigmented colonies. They were found to grow optimally at 37 °C, at pH 7.0 and in the presence of 2.6 M NaCl and 0.05 M Mg2?. The major polar lipids were identified as those typical for members of the Halobacteriaceae but also included major glycolipids chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-1), mannosyl glucosyl diether (DGD-1) and two unidentified ones. The 16S rRNA gene sequences of the three strains were 99.8–100 % identical, showing most similarity to sequences of members of the family Halobacteriaceae, and clustering together as a distinct clade in phylogenetic tree reconstructions. The rpoB0 gene similarities between the three strains were 98.7–100 % and lower to the sequences of other halobacteria. Their DNA G?C contents were determined to be 65.1–65.5 mol%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strains XD46T (=CGMCC 1.12237T =
Electronic supplementary material The online version of this article (doi:10.1007/s10482-013-0061-8) contains supplementary material, which is available to authorized users. H.-L. Cui (&) X.-X. Qiu School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Jingkou District, Zhenjiang 212013, People’s Republic of China e-mail: [email protected]
JCM 18649T), YJ-63-S1 (=CGMCC 1.12574) and ZS1-H (=CGMCC 1.12544) represent a novel species in a new genus within the family Halobacteriaceae, for which the name Salinarubrum litoreum gen. nov., sp. nov. is proposed. Keywords Salinarubrum litoreum gen. nov., sp. nov. Halophilic archaea Marine solar saltern
Introduction Marine solar salterns are common artificial habitats for diverse halophilic archaea, members of the family Halobacteriaceae within the order Halobacteriales (Oren 2006). In recent years, from this kind of hypersaline environment, more and more remarkable isolates representing novel genera have been cultivated, described and proposed, such as Halonotius pteroides (Burns et al. 2010), Halogranum rubrum, Halopelagius inordinatus, Halorussus rarus, Halolamina pelagica, Halobellus clavatus, Halorientalis regularis, Halorubellus salinus (Cui et al. 2010a, b, c, 2011a, b, 2012), Natronoarchaeum mannanilyticum, Salarchaeum japonicum (Shimane et al. 2010, 2011), Halarchaeum acidiphilum (Minegishi et al. 2010a), Halomarina oriensis (Inoue et al. 2011), Halohasta litorea (Mou et al. 2012) and Halomicroarcula pellucida (Echigo et al. 2013). This rapid expansion in new taxa indicates that the members of family
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Halobacteriaceae are more diverse than was previously recognized. During our surveys on halophilic archaeal diversity of marine solar salterns of Eastern and Southern China, three haloarchaeal strains, XD46T, YJ-63-S1 and ZS-1-H, showing high similarity of 16S rRNA gene sequence, were isolated from three different marine solar salterns. In this study, we characterize these three strains and propose they represent a novel species of a novel genus, Salinarubrum.
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to the proposed minimal standards for description of new taxa in the order Halobacteriales (Oren et al. 1997). The type strains Halorussus rarus TBN4T, Halalkalicoccus jeotgali JCM 14584T, Halorubellus salinus GX3T, Haladaptatus paucihalophilus JCM 13897T were selected as reference strains in phenotypic tests. These reference strains were obtained from China General Microbiological Culture Collection and Japan Collection of Microorganisms. Chemotaxonomic characterization
Materials and methods Isolation and cultivation of halophilic archaeal strains Strain XD46T was isolated from a surface brine sample taken from Xidi marine solar saltern, at Xiamen, Fujian Province, China (24°330 5100 N, 118°180 2100 E; elevation, sea level) in 2010. Strain YJ-63-S1 was isolated from a sediment sampling from Yangjiang marine solar saltern, at Yangjiang, Guangxi Province, China (21°310 4800 N, 111°280 500 E; elevation, sea level) in 2012. Strain ZS-1-H was isolated from the sediment of Zhoushan marine solar saltern, at Zhoushan, Zhejiang Province, China (29°560 5600 N, 122°200 2000 E; elevation, sea level) in 2012. The neutral oligotrophic haloarchaeal medium (NOM) was used for the isolation procedure, and contained the following ingredients (g/L): yeast extract (Oxoid) 0.05, fish peptone (Sinopharm Chemical Reagent Co., Ltd.) 0.25, sodium pyruvate 1.0, KCl 5.4, K2HPO4 0.3, CaCl2 0.25, NH4Cl 0.25, MgSO47H2O 26.8, MgCl26H2O 23.0, NaCl 184.0 (pH adjusted to 7.0–7.2 with 1 M NaOH solution). The medium was solidified with 2.0 % agar. The strains were routinely grown aerobically at 37 °C in NOM-3 medium (NOM series medium) with the following modifications (g/L): yeast extract 1.0, fish peptone 0.25, sodium formate 0.25, sodium acetate 0.25, sodium lactate 0.25 and sodium pyruvate 0.25. Phenotypic determination Determination of morphology and growth characteristics, nutrition, miscellaneous biochemical tests and sensitivity to antimicrobial agents were performed for all species in the same basic medium, NOM, according
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Polar lipids were extracted using a chloroform/methanol system and analysed using one-and two-dimensional TLC, as described previously (Cui et al. 2010b). Merck silica gel 60 F254 aluminium-backed thin-layer plates were used for TLC analyses. In two-dimensional TLC, the first solvent was chloroform–methanol–water (65:25:4, by vol.) and the second solvent was chloroform–methanol–acetic acid–water (80:12:15:4, by vol.). The latter solvent mixture was also used in onedimensional TLC. Two specific detection spray reagents, phosphate stain reagent for phospholipids and a-naphthol strain for glycolipids, were used. The general detection reagent, sulfuric acid–ethanol (1:2, by vol.), was also used to detect total polar lipids. The presence of phospholipids and glycolipids on the twodimensional TLC was confirmed by comparing with one-dimensional TLC on which the polar lipid profiles of reference strains were developed. Phylogenetic and genomic analysis Genomic DNA from halophilic archaeal strains was prepared as described previously (Cui et al. 2011c). The 16S rRNA genes were amplified, cloned and sequenced according to the previous protocol (Cui et al. 2009). PCR-mediated amplification and sequencing of the rpoB0 genes were performed as described previously (Minegishi et al. 2010b). Multiple sequence alignments were performed using the ClustalW program integrated in the MEGA 5 software (http://www.megasoftware. net/). Phylogenetic trees were reconstructed using neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) algorithms in the MEGA 5 software (Tamura et al. 2011). Gene sequence similarity among halophilic archaea was calculated using the Pairwise-Distance computing function of MEGA 5. The DNA G?C content was determined from
Antonie van Leeuwenhoek (2014) 105:135–141
the mid-point value (Tm) of the thermal denaturation method (Marmur and Doty 1962) at 260 nm with a Beckman-Coulter DU800TM spectrophotometer equipped with a high-performance temperature controller. Halomicrobium mukohataei JCM 9738T was selected as reference strain for these analyses, and the formula G?C mol% unknown strain = G?C mol% reference strain ? 2.08 9 (Tm unknown strain - Tm reference strain) was used to calculate the G?C content from the known Tm value (Owen and Pitcher 1985).
Results and discussion Cells of strains XD46T, YJ-63-S1 and ZS-1-H were observed to be motile and pleomorphic when grown in NOM-3 liquid medium (Supplementary Fig. S1). They were observed to stain Gram-negative and their colonies were observed to be red-pigmented. The three strains were found to be able to grow at 20–50 °C (optimum 37 °C), in the presence of 0.9–4.8 M NaCl (optimum 2.6 M NaCl), with 0.005–1.0 M MgCl2 (optimum 0.05 M MgCl2) and at pH 5.0–9.0 (optimum pH 7.0). The cells of all isolates were observed to lyse in distilled water and the minimum NaCl concentration that prevented cell lysis was 50 g/L. The three strains did not grow under anaerobic conditions using nitrate, DMSO or L-arginine, but nitrate reduction to nitrite was observed. They were found to produce H2S from sodium thiosulfate but did not produce indole from tryptophan. They hydrolyzed gelatin, Tween 80 and casein, but did not hydrolyze starch. The main phenotypic characteristics differentiating strains XD46T, YJ63-S1 and ZS-1-H from the related members of family Halobacteriaceae were: cell motility, cell lysis in distilled water, optimum NaCl for growth, the growth requirement for Mg2?, optimum pH, utilization of specific carbon sources, indole formation, starch, casein gelatin, Tween 80 and hydrolysis (Table 1). More detailed results of phenotypic tests and nutritional features of these strains are given in the species descriptions. The major polar lipids of the three strains were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, two major glycolipids chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-1) and mannosyl glucosyl diether (DGD-1), respectively. Three unidentified lipids were also detected (Supplementary Fig. S2).
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The polar lipid profile set strains XD46T, YJ-63-S1 and ZS-1-H apart from members of the genus Halorussus which contain sulfated galactosyl mannosyl glucosyl diether (S-TGD-1), S-DGD-1, galactosyl mannosyl glucosyl diether, DGD-1 and an unknown diglycosyl diether (Cui et al. 2010b), members of the genus Halorubellus which contain S-TGD-1, S-DGD1, DGD-1 and an unidentified lipid (Cui et al. 2012), and members of the Halalkalicoccus which contain trace unidentified glycolipids (Roh et al. 2007) (Supplementary Fig. S2). Eight complete 16S rRNA gene sequences of each strain were obtained (1,475 bp in length), and these indicated that each strain has a single 16S rRNA gene sequence. They shared 99.8–100 % sequence identities, and all three strains showed low levels of 16S rRNA gene sequence similarity to other members of the family Halobacteriaceae. The closest related recognized species were Halalkalicoccus jeotgali (92.3–92.4 % similarity), Halalkalicoccus tibetensis (92.2–92.3 % similarity), Halorussus rarus (91.6–91.9 % similarity) and Halorubellus salinus (90.4–91.9 % similarity). Phylogenetic tree reconstructions using the NJ algorithm revealed that strains XD46T, YJ-63-S1 and ZS-1-H tightly clustered with each other forming a distinct clade (Fig. 1a), separate from other recognized genera of the Halobacteriaceae. The closest branching species to this clade was Halorussus rarus TBN4T. Their phylogenetic position was also confirmed in other trees generated using the MP and ML algorithms (data not shown). The rpoB0 genes of all the three strains were sequenced and found to be identical in length (1,827 bp) and their sequence identities were 98.7–100 %. The most closely similar sequence was the corresponding gene of Halorussus rarus TBN4T, but the level of similarity to the three isolates was much lower (85.8–85.9 %). In phylogenetic tree reconstructions using rpoB0 gene sequences, strains XD46T, YJ-63-S1 and ZS-1-H clustered tightly with each other, forming a distinct clade, this clade branched nearby members of Halalkalicoccus and Halorussus (Fig. 1b). The phylogenetic position was also confirmed in trees generated using the MP and ML algorithms (data not shown). The DNA G?C content of strains XD46T, YJ-63S1 and ZS-1-H was determined to be 65.5, 65.1 and 65.1 mol%, respectively. These values are higher than those of members of the genera Haladaptatus (54.0–59.2 mol%) (Cui et al. 2010d) and
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Table 1 Differential characteristics between strain XD46T, strain YJ-63-S, strain ZS-1-H and closely related members of the family Halobacteriaceae Characteristic
1
2
3
4
5
6
7
Motility
?
?
?
?
±
–
?
Cells lyse in distilled water
?
?
?
?
–
–
?
Optimum NaCl (M)
2.6
2.6
2.6
2.1
2.6-3.4
2.6-3.4
3.1
Mg2? required
?
–
–
–
?
–
–
Optimum pH
7.0
7.0
7.0
7.0
7.0
7.0/9.0
7.0
D-mannose
?
?
?
?
±
–
–
D-galactose
?
?
?
?
±
–
±
D-fructose
–
–
–
–
±
?
–
Maltose
–
–
–
?
±
?
–
Lactose
–
–
–
?
±
?
– –
Utilization of:
Indole formation
–
–
–
?
?
±
Starch hydrolysis
–
–
–
?
±
–
–
Gelatin hydrolysis
?
?
?
?
?
–
?
Casein hydrolysis Tween 80 hydrolysis
? ?
? ?
? ?
? ?
± ?
– –
? –
Presence of PGS
–
–
–
?
?
–
–
G?C content (mol%)
65.5
65.1
65.1
65.4-66.1
54.0-59.2
61.5-63.2
67.2-67.3
Taxa: 1, XD46T; 2, YJ-63-S; 3, ZS-1-H; 4, Halorussus; 5, Haladaptatus; 6, Halalkalicoccus; 7, Halorubellus. Symbols: ?, positive; –, negative. All data from the present study
Halalkalicoccus (61.5–63.2 mol%) (Roh et al. 2007), lower than those of members of the genus Halorubellus (67.2–67.3 mol%) (Cui et al. 2012) and close to the values for members of the genus Halorussus (65.4–66.1 mol%) (Cui et al. 2010b). This polyphasic taxonomic study provides evidence that strains XD46T, YJ-63-S1 and ZS-1-H represent a novel species of a new genus within the family Halobacteriaceae, for which the name Salinarubrum litoreum gen. nov., sp. nov. is proposed. The type strain is XD46T (= CGMCC 1.12237T = JCM 18649T) and the reference strains are YJ-63-S1 (= CGMCC 1.12574) and ZS-1-H (= CGMCC 1.12544). Characteristics that distinguish strains XD46T, YJ-63-S1 and ZS-1-H from other genera within the family Halobacteriaceae are shown in Table 1.
Cells lyse in distilled water. Catalase test is positive and the oxidase test is negative. Extremely halophilic, with growth occurring in media containing 0.9–4.8 M NaCl; most strains grow best at 2.6 M NaCl. The optimum magnesium concentration is 0.05 M. Temperatures between 20 and 50 °C and pH between 5.0 and 9.0 may support growth. Sugars are metabolized, in some cases with the formation of acids. The polar lipids are phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, two major glycolipids chromatographically identical to S-DGD-1 (sulfated mannosyl glucosyl diether) and DGD-1 (mannosyl glucosyl diether), respectively. Three unidentified lipids are also present. The genomic DNA G?C content is between 65.1 and 65.5 mol%. Isolated from marine solar salterns. The type species is Salinarubrum litoreum. Recommended three-letter abbreviation: Srr.
Description of Salinarubrum gen. nov. Description of Salinarubrum litoreum sp. nov. Salinarubrum (Sa.li.na.ru’brum. L. fem. pl. n. salinae salterns, salt works; L. neut. adj. rubrum red; M.L. neut. n. Salinarubrum the red archaeon from salt works). Cells are pleomorphic under optimal growth conditions, stain Gram-negative and are aerobic heterotrophs.
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Salinarubrum litoreum (li.to’re.um. L. neut. adj. litoreum, of or belonging to the sea-shore). Cells are motile, pleomorphic-shaped under optimal growth conditions and stain Gram-negative. Colonies
Antonie van Leeuwenhoek (2014) 105:135–141
a
139 Haladaptatus litoreus RO1-28T (EU887285)
100
Haladaptatus paucihalophilus JCM 13897T (HM159609)
57
Halorubellus salinus GX3T rrnA (GU951429) Halorubellus salinus GX3T rrnB (HQ236376)
100
Halorussus rarus TBN4T (GQ282618)
0.02
Strain XD46T (JQ237118)
72 84
100
Strain YJ-63-S (KC918824)
65 Strain
ZS-1-H (KC918825)
Halalkalicoccus jeotgali B3T (NC_014297)
100
Halalkalicoccus tibetensis DS12T (AF435112) Halococcus morrhuae ATCC 17082T (X00662)
98
Halococcus salifodinae DSM 8989T (AB004877)
100
Natronomonas pharaonis DSM 2160T (NC_007426) Methanospirillum hungatei JF-1T (NC_007796)
b
100 100
Strain YJ-63-S (KF316330) Strain ZS-1-H (KF316331) Strain XD46T (KF316332)
63
Halalkalicoccus jeotgali JCM 14584T (AB477140) 100
0.05
Halalkalicoccus tibetensis JCM 11890T (AB477141) Halorussus rarus TBN4T (JN120805)
52
Halorubellus salinus GX3T (JN164652) Haladaptatus litoreus RO1-28T (JN315801)
90
100
Haladaptatus paucihalophilus JCM 13897T (AB477139) Halococcus morrhuae JCM 8876T (AB477157)
96
Halococcus salifodinae JCM 9578T (AB477160) Natronomonas pharaonis DSM 2160T (NC_007426) Methanospirillum hungatei JF-1T (NC_007796)
Fig. 1 Neighbour-joining phylogenetic tree reconstructions based on 16S rRNA gene (a) and rpoB0 gene (b) sequences, showing the relationships between the isolates and related members within the family Halobacteriaceae. Bootstrap values
(%) are based on 1,000 replicates and are shown for branches with more 50 % bootstrap support. Bar represents expected changes per site
on agar plates containing 2.6 M NaCl are red, elevated and round. Chemoorganotrophic and aerobic. Growth occurs at 20–50 °C (optimum 37 °C), at 0.9–4.8 M NaCl (optimum 2.6 M), at 0.005–1 M MgCl2 (optimum 0.05 M) and at pH 5.0–9.0 (optimum pH 7.0). Cells lyse in distilled water and the minimal NaCl concentration to prevent cell lysis is 50 g/L. Catalasepositive and oxidase-negative. Does not grow under anaerobic conditions with nitrate, arginine or DMSO. Nitrate reduction to nitrite is observed but gas formation from nitrate is not. H2S is produced from sodium thiosulfate. Indole formation is negative. Hydrolyzes casein, gelatin and Tween 80 but does not hydrolyze
starch. The following substrates are utilized as single carbon and energy sources for growth: citrate, fumarate, D-glucose, glycerol, DL-lactate, L-malate, pyruvate, Dsorbitol and sucrose. L-glutamate is utilized as single carbon, nitrogen or energy sources for growth. No growth occurs on acetate, L-alanine, L-arginine, Laspartate, D-fructose, D-galactose, glycine, lactose, Llysine, maltose, D-mannitol, D-mannose, L-ornithine, Dribose, L-sorbose, starch, succinate or D-xylose. Acid is produced from D-glucose and sucrose. Sensitive to the following antimicrobial compounds (lg per disc, unless otherwise indicated): bacitracin (0.04 IU per disc), ciprofloxacin (5), mycostatin (100), nitrofurantoin
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(300), norfloxacin (10) and novobiocin (30). Resistant to the following antimicrobial compounds: ampicillin (10), chloramphenicol (30), erythromycin (15), gentamicin (10), kanamycin (30), nalidixic acid (30), neomycin (30), penicillin G (10 IU per disc), rifampin (5), streptomycin (10), tetracycline (30), trimethoprim (5) and vancomycin (30). The polar lipids are phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, two major glycolipids chromatographically identical to S-DGD-1 and DGD-1, respectively. Three unidentified lipids are also detected. The DNA G?C content of strain XD46T is 65.5 mol% (Tm). The type strain is XD46T (=CGMCC 1.12237T = JCM 18649T) and was isolated from Xidi marine solar saltern at Xiamen, Fujian Province, China. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains XD46T, YJ63-S1 and ZS-1-H are JQ237118, KC918824 and KC918825 respectively. Those for the rpoB0 gene sequences of strains XD46T, YJ-63-S1 and ZS-1-H are KF316330, KF316331 and KF316332, respectively. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31370054), the grant from China Ocean Mineral Resources R&D Association (COMRA) Special Foundation (DY125-15-R-03), the Qinglan Project of Jiangsu Province and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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